This is the competing renewal for CA548o7 Objectives during the past cycle of this project included the determination of a) autocrine TGF alpha function and b) mechanisms controlling TGF alpha expression in colon cancers. Accomplishments have included the determination that autocrine TGF alpha: in both early and late stage colon cancers functions primarily in the process of re-entering the cycling state from environmental conditions which limit cell growth (e.g.. growth factor deprivation) and does not appear to have a significant role in actively cycling populations (e.g., exponential growth in tissue culture). Late stage colon cancer cell lines differ from early stage cells in that they are completely independent of exogenous growth factors for re-initiation of DNA synthesis from growth arrest whereas early stage cells require an exogenous insulin-like growth factor l receptor (IGFIR) ligand for cell cycle re-entry. We have found that the growth factor independence of late stage malignant cells arises from inappropriate TGF alpha: expression (not overexpression) and EGF receptor (EGFR) activation in growth arrested cells. This has the effect of producing constitutive EGFR activation, an event long associated with transformation. Early stage cells do not express TGF ALPHA: or show significant EGFR activation at growth arrest. IGFIR activation in early stage cells regenerates their autocrine TGF ALPHA: expression and EGFR activation events which we have shown to be necessary for DNA synthesis in these cells. Finally, we have found that autocrine TGF~ represses autocrine TGF~ expression in early stage cells. Inhibition of TGF~ function leads to enhanced expression of autocrine TGF ALPHA: and EGFR activation in growth arrested cells along with the acquisition of independence from exogenous growth factors and malignant progression to a late stage phenotype. Exogenous TGFj3 treatment also blocks IGFIR mediated induction of TGF ALPHA: mRNA in early stage cells. Several issues arise from these results. The first issue we will address is the mechanism by which IGFIR activation stimulates TGF ALPHA: re- expression from the growth arrested state. The second issue that will be addressed in the renewal project is the mechanism by which TGFj3 suppresses TGF alpha expression. The results from the last cycle of the project have shown that inappropriate expression of TGF alpha provides growth factor independence, but they have not addressed how inappropriate TGF alpha expression carries this out. Therefore, this issue will also be addressed in the renewal project.